| BackgroundUlcerative colitis(UC)is a major form of inflammatory bowel disease with a clinical manifestation of bloody stool,abdominal pain,and diarrhea.The burden of UC is increasing worldwide,especially in developing countries.Many factors have been reported to be involved in UC,including genetic susceptibility,immune system dysregulation,and diet.However,understanding of the pathology of UC remains inadequate.Enterochromaffin(EC)cells are a major component of the enteroendocrine system.Although they only constitute about 1%of the intestinal epithelium,EC cells play a crucial role in gut homeostasis through receiving stimuli from the gastrointestinal tract,producing 5-HT and various other enteroendocrine hormones.Previous studies have also reported that UC is associated with a rise in the number of EC cells;however,the exact role of EC cells in UC is still unknown.The scarcity and scattered distribution of EC cells has severely limited the investigation of their function using currently available methods.Previous studies of EC cells in UC patients have mainly focused on enhancement of the secretory function,particularly with regard to 5HT and chromogranin A,disregarding other aspects of EC cell function.In recent years,the rapid progression in technologies such as fluorescence-activated cell sorting and single-cell RNA sequencing has enabled transcriptomic analyses of EC cells in human intestinal organoids and in mice;however,knowledge of the biology of human EC cells in situ remains inadequate.Although several recent studies have involved single-cell RNA-seq of colonoscopic biopsy specimens from UC patients,these studies have produced few findings regarding EC cells,probably due to limited sequencing depth and EC cell scarcity.A novel technology termed nanoString GeoMX DSP has provided new opportunities to the research of EC cells,which can perform transcriptive sequencing on FFPE samples with high spatial selectivity.Here in this study,we involved multiple transcriptomic methods including nanoString GeoMX DSP,scRNA-seq and bulk RNA-seq to investigate the molecular expression patterns in EC cells from colonoscopic biopsy specimens from both UC patients and healthy controls,as well as to explore its possible impacts on the pathology of ulcerative colitis.Part Ⅰ:The transcriptomic profiling of human enterochromaffin cells via nanoString GeoMX DSPAims:As a major component of the enteroendocrine system,EC cells play a key role in gut homeostasis.However,the scarcity of EC cells has limited the investigation of their function technically.In this study,we applied a novel technology termed nanoString GeoMX DSP to profile the transcriptomic pattern of human EC cells.Methods:A total of 78 biopsy samples was collected,screened for the number of EC cells via immunohistochemistry and constructed on to tissue microarrays for nanoString GeoMX DSP experiment.Transcriptomic data from EC cells and background epithelium were collected,respectively.After quality control,average target count and limit of quantitation was calculated.Principal component analysis was performed.The expression level of TPH1 and CHGA in each group was measured.Results:About 1,8000 targets from 19 ROIs were sequenced.The median number of targets above LOQ was 3,267,varied from 1,355 to 7,267.Principal component analysis indicates difference in gene expression between groups.The expression of THP1 and CHGA was significantly increased in EC groups.Conclusions:Our results suggest the capability of nanoString GeoMX DSP for the profiling of the transcriptomic pattern of human EC cells.NanoString GeoMX DSP is a novel and reliable method for EC cell research.Part Ⅱ:NanoString GeoMX DSP and scRNA-seq revealed novel markers in human enterochromaffin cellsAims:EC cells play a key role in gut homeostasis.However,the scarcity of EC cells and a lack of EC markers have limited the investigation of their function.In this study,we profiled the transcriptomic pattern of human EC cells using nanoString GeoMX DSP and single cell RNAseq data.Methods:Differential expression analysis and weighted gene co-expression network analysis were performed to identify the EC-related genes.Single-cell transcriptomic data for colonic cells from 18 UC patients and 12 healthy individuals were downloaded from Single Cell Portal.Standard Seurat procedure was applied for data processing and clustering.EC cell was identified as CHGAhigh TPH1high PCSK1Nhigh PYYlow SCGlow.Differential expression analysis was performed.The results were compared with those from nanoString GeoMX DSP.Validation through immunofluorescent staining was performed.Results:In the DSP data,differential expression analysis found 10 genes significantly enriched in EC cells compared to background epithelium.A co-expression network containing 17 hub genes,including TPH1,CHGA,and GCLC,was identified in EC cells.By comparing the results from DSP and single cell RNA-seq,RGS2,PCSK1N,CHGB,CHGA,TPH1,CRYBA2,SCG5,IGFBP3,and DDC were identified as EC-related markers.Apart from TPH1,RGS2 also showed high specificity to EC cells.The co-localization between RGS2 and CHGB,and EC cells was further confirmed by immunofluorescence staining.Conclusions:Our results illuminate the distinctive transcriptional signatures of EC cells in human colon.Novel markers,such as RGS2,CHGB and CRYBA2,were identified in EC cells,whose function in gut homeostasis remains unknown.Part Ⅲ:The profiling of the mechanism of enterochromaffin cells in the pathology of ulcerative colitis via nanoString GeoMX DSPAims:EC cells participate in maintaining gut homeostasis via various mechanisms.However,previous studies regarding the role of EC cells in ulcerative colitis are largely restricted in the elevated level of 5-HT and CHGA.Understanding of the biological behavior of EC cells under the condition of ulcerative colitis remained inadequate.Methods:To determine the transcriptomic changes of EC cells in UC,differential expression analysis was performed using both DSP and single cell RNA-seq data,followed by RT-qPCR to confirm the results.The differentially expressed genes between the group EC-Con and Epi-Con,and between EC-UC and EC-con,underwent the process of gene set enrichment analysis using GO and KEGG gene set.The proportion of EC cells in UC and control was calculated.Transcriptive factor enrichment analysis was performed using ChEA3.Bulk RNA-seq data of UC patients was downloaded from Gene Expression Omnibus.Correlation between EC-related genes and markers regarding barrier dysfunction in UC was performed.Results:Compared to healthy control,49 genes were found up-regulated in EC cells from UC patients,among which the elevated expression level of CHGB in UC was confirmed by both single cell RNA-seq data and qRT-PCR.In healthy individuals,EC cells are enriched in functionally concentrated in protein and bioamine synthesis,such as "protein targeting to ER","nuclear-transcribed mRNA catabolic process",and "translation initiation".In UC patients,EC cells gained increased capacity for protein synthesis,along with novel immunological functions such as antigen processing and presentation,whereas chemical sensation was down-regulated.Compared to healthy controls,non-inflamed mucosa from UC patients showed an increased proportion of EC cells.Several transcriptive factors,such as MYRFL,IRF9,ISX,IRF7 and BATF2,were associated with the changes in the EC cells of UC.ISX and ATOH2 was related in the upregulation of CHGB in ulcerative colitis.CHGB,GCLC,DDC and RGS2 were highly correlated with barrier marker CLDN2.Conclusions:Compared with background epithelium,EC cells are adapted for protein synthesis and secretion in healthy colon.EC cells’ newly observed functional shift from sensation to secretion and immunity indicate their pivotal role in UC.Through the regulation of ISX and ATOH2,the secretion of CHGB in EC cells in UC was elevated and was highly specifically correlated with CLDN2.The dramatic rise of the proportion of EC cell in UC indicates a crucial role in maintaining barrier function and gut homeostasis. |
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